番鸭细小病毒样颗粒的制备与鉴定
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摘要
为在昆虫细胞中表达番鸭细小病毒主要结构蛋白VP3,并探讨重组蛋白VP3能否在昆虫细胞中自动组装成病毒样颗粒,高保真扩增VP3基因,克隆入杆状病毒转移载体pFastBac1中,获得重组转移质粒pFB-VP3,转化DH10Bac感受态细胞,经抗性、蓝白斑筛选及PCR鉴定,得到重组Bacmid DNA。将其转染昆虫细胞Sf9,制备重组杆状病毒rBac-VP3,利用SDS-PAGE、Western-blot、间接免疫荧光试验对重组蛋白的表达情况进行检测,负染电镜观察病毒样颗粒,并通过Western-blot对病毒样颗粒进行鉴定。SDS-PAGE和Western-blot结果表明重组蛋白大小正确。间接免疫荧光试验结果显示,重组蛋白获得了成功表达。利用负染电镜可观察到直径约20 nm的典型二十面体病毒样颗粒。Western-blot结果显示该病毒样颗粒由VP3蛋白组成。本研究首次成功利用杆状病毒/昆虫细胞表达系统制备了番鸭细小病毒样颗粒,为番鸭细小病毒病新型基因工程疫苗的研制奠定了基础。
In order to express the structural protein VP3 of Muscovy duck parvovirus(MDPV) with the baculovirus/insect cell expression system and to explore whether the recombinant proteins expressed in insect cells could self-assemble into virus-like particles(VLPs),the structural gene VP3 was amplified by PCR and cloned into the baculovirus expression vector p Fast Bac1,and the resultant plasmid was named p FB-VP3.Then the plasmid p FB-VP3 was transformed into the competent cells DH10 Bac,and the positive recombinant bacmid DNA was screened through the resistant and the blue-white plague screening and identified with PCR.The recombinant baculovirus r Bac-VP3 was obtained by transfection the insect cells Sf9 with the recombinant bacmid DNA.The expression of the recombinant proteins was identified with SDS-PAGE,Western-blot and indirect immunofluorescence assay(IFA),VLPs were certified by negative staining electron microscopy,and the structure of VLPs was analyzed by Western-blot.SDS-PAGE and Westernblot assay revealed that the size of the recombinant proteins was correct,the results of IFA indicated that the recombinant proteins were expressed successfully in insect cells.The results of negative staining electron microscopy showed the typical icosahedral particles with a diameter of 20 nm approximately.Western-blot results indicated that the VLPs were composed of the structural protein VP3.MDPVlike particles were first prepared with baculovirus/insect cell expression system,which laid the foundation for the development of a new genetic engineering vaccine for Muscovy duck parvovirus infection.
In order to express the structural protein VP3 of Muscovy duck parvovirus(MDPV) with the baculovirus/insect cell expression system and to explore whether the recombinant proteins expressed in insect cells could self-assemble into virus-like particles(VLPs),the structural gene VP3 was amplified by PCR and cloned into the baculovirus expression vector p Fast Bac1,and the resultant plasmid was named p FB-VP3.Then the plasmid p FB-VP3 was transformed into the competent cells DH10 Bac,and the positive recombinant bacmid DNA was screened through the resistant and the blue-white plague screening and identified with PCR.The recombinant baculovirus r Bac-VP3 was obtained by transfection the insect cells Sf9 with the recombinant bacmid DNA.The expression of the recombinant proteins was identified with SDS-PAGE,Western-blot and indirect immunofluorescence assay(IFA),VLPs were certified by negative staining electron microscopy,and the structure of VLPs was analyzed by Western-blot.SDS-PAGE and Westernblot assay revealed that the size of the recombinant proteins was correct,the results of IFA indicated that the recombinant proteins were expressed successfully in insect cells.The results of negative staining electron microscopy showed the typical icosahedral particles with a diameter of 20 nm approximately.Western-blot results indicated that the VLPs were composed of the structural protein VP3.MDPVlike particles were first prepared with baculovirus/insect cell expression system,which laid the foundation for the development of a new genetic engineering vaccine for Muscovy duck parvovirus infection.
引文
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[14]邹小辉,董流昕,宋敬东,等.人细小病毒B19病毒样颗粒的制备[J].生物工程学报,2009,25(4):575-579.ZOU Xiao-hui,DONG Liu-xin,SONG Jing-dong,et al.Preparation of human parvovirus B19 virus-like particles[J].Chinese Journal of Biotechnology,2009,25(4):575-579.(in Chinese)
[15] GHOSH S,PARVEZ M K,BANERJEE K,et al.Baculovirus as mammalian cell expression vector for gene therapy:an emerging strategy[J].Molecular Therapy,2002,6(1):5-11.
[16] MARANGA L,CRUZ P E,AUNINS J G,et al.Production of core and virus-like particles with baculovirus infected insect cells[J].Advances in Biochemical Engineering/Biotechnology,2002,74:183-206.
[17] KU Z,LIU Q,YE X,et al.A virus-like particle based bivalent vaccine confers dual protection against enterovirus 71 and coxsackie virus A16 infections in mice[J].Vaccine,2014,32(34):4296-4303.
[18] LIN Y L,YU C I,HU Y C,et al.Enterovirus type 71neutralizing antibodies in the serum of macaque monkeys immunized with EV71 virus-like particles[J].Vaccine,2012,30(7):1305-1312.
[19] PALOMARES L A,RAMIREZ O T.Challenges for the production of virus-like particles in insect cells:The case of rotavirus-like particles[J].Biochemical Engineering Journal,2009,45(3):158-167.
[2]余兵,王永坤,朱国强,等.番鸭细小病毒M91G27株病毒结构蛋白VP3编码基因的克隆与鉴定[J].中国预防兽医学报,2000,22(6):408-411.YU Bing,WANG Yong-kun,ZHU Guo-qiang,et al.Cloning and identification of VP3-coding gene of M91G27strain of Muscovy parvovirus[J].Chinese Journal of Preventive Veterinary Medicine,2000,22(6):408-411.(in Chinese)
[3] POONIA B,JAROSINAKI K W,SCHAT K A,et al.Isolation and molecular characterization of a new Muscovy duck parvovirus from Muscovy ducks in the USA[J].Avian Pathology,2006,35(6):435-441.
[4] CHACKERIAN B.Virus-like particles:Flexible platforms for vaccine development[J].Expert Review of Vaccines,2007,6(3):381-390.
[5] BUONAGURO L,TAGLIAMONTE L,TORNESELLO M L,et al.Developments in virus-like particle-based vaccines for infectious diseases and cancer[J].Expert Review of Vaccines,2011,10(11):1569-1583.
[6] KUSHNIR N,STREATFIELD S J,YUSIBOV V.Virus-like particles as a highly efficient vaccine platform:diversity of targets and production systems and advances in clinical development[J].Vaccine,2012,31(1):58-83.
[7] JAIN N K,SAAHNI N,KUMRU O S,et al.Formulation and stabilization of recombinant protein based viruslike particle vaccines[J].Advanced Drug Delivery Reviews,2015,93:42-55.
[8] BRIGHT R A,CARTER D M,DANILUK S,et al.Influenza virus-like particles elicit broader immune responses than whole virion inactivated influenza virus or recombinant hemagglutinin[J].Vaccine,2007,25(19):3871-3878.
[9] SCHELLENBACHER C,RODEN R,KIRNBAUER R.Chimeric L1-L2 virus-like particles as potential broadspectrum human papillomavirus vaccines[J].Journal of Virology,2009,83(19):10085-10095.
[10] THOMSEN D R,ROOF L L,HOMA F L.Assembly of herpes simplex virus(HSV)intermediate capsids in insect cells infected with recombinant baculoviruses expressing HSV capsid proteins[J].Journal of Virology,1994,68(4):2442-2457.
[11] SOMASUNDARAM B,CHANG C,FAN Y Y,et al.Characterizing enterovirus 71 and Coxsackie virus A 16 virus-like particles production in insect cells[J].Methods,2016,95:38-45.
[12] JU H Y,WEI N,WANG Q,et al.Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose[J].Biochem ical and Biophysical Research Communications,2011,409(1):131-136.
[13] LE GALL-RECULEG,JESTIN V,CHAGNAUD P,et al.Expression of Muscovy duck parvovirus capsid proteins(VP2 and VP3)in a baculovirus expression system and demonstration of immunity induced by the recombinant proteins[J].Journal of General Virology,1996,77(3):2159-2163.
[14]邹小辉,董流昕,宋敬东,等.人细小病毒B19病毒样颗粒的制备[J].生物工程学报,2009,25(4):575-579.ZOU Xiao-hui,DONG Liu-xin,SONG Jing-dong,et al.Preparation of human parvovirus B19 virus-like particles[J].Chinese Journal of Biotechnology,2009,25(4):575-579.(in Chinese)
[15] GHOSH S,PARVEZ M K,BANERJEE K,et al.Baculovirus as mammalian cell expression vector for gene therapy:an emerging strategy[J].Molecular Therapy,2002,6(1):5-11.
[16] MARANGA L,CRUZ P E,AUNINS J G,et al.Production of core and virus-like particles with baculovirus infected insect cells[J].Advances in Biochemical Engineering/Biotechnology,2002,74:183-206.
[17] KU Z,LIU Q,YE X,et al.A virus-like particle based bivalent vaccine confers dual protection against enterovirus 71 and coxsackie virus A16 infections in mice[J].Vaccine,2014,32(34):4296-4303.
[18] LIN Y L,YU C I,HU Y C,et al.Enterovirus type 71neutralizing antibodies in the serum of macaque monkeys immunized with EV71 virus-like particles[J].Vaccine,2012,30(7):1305-1312.
[19] PALOMARES L A,RAMIREZ O T.Challenges for the production of virus-like particles in insect cells:The case of rotavirus-like particles[J].Biochemical Engineering Journal,2009,45(3):158-167.
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